Crystal Structure of the Dimerized N Terminus of Porcine Circovirus Type 2 Replicase Protein Reveals a Novel Antiviral Interface.
Crystal Structure of the Dimerized N Terminus of Porcine Circovirus Type 2 Replicase Protein Reveals a Novel Antiviral Interface.
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猪圆环病毒 2 型复制酶蛋白二聚化 N 末端的晶体结构揭示了一种新型抗病毒界面。
DOI：
10.1128/jvi.00724-18
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发表时间：
2018
影响因子：
5.4
通讯作者：
Song Yunfeng
中科院分区：
文献类型：
--
作者：
Luo Gao;Zhu Xiongfei;Lv Yang;Lv Bomin;Fang Jin'e;Cao Shengbo;Chen Huanchun;Peng Guiqing;Song Yunfeng
猪圆环病毒（PCV） ORF1编码两个复制酶（Rep）蛋白：Rep和Rep′；Rep是完整的ORF1转录本，Rep’是通过剪接产生的截断转录本。这两种蛋白对PCV的滚圈复制（RCR）至关重要。Rep和Rep '的n端序列相同，相互作用形成同源或异源二聚体。三种类型的二聚体在复制过程中执行不同的功能。未对连接端进行结构检查。在本研究中，二聚Rep蛋白N末端的晶体结构在2.7 Å被分解。二聚体蛋白由9个分子间氢键和15对疏水相互作用维持。氨基酸残基Ile37参与了11种疏水相互作用，主要是通过它的侧链。为了找到蛋白质二聚化和病毒复制的优势位点，通过丙氨酸取代产生了一系列突变蛋白和病毒复制子。在所有的单氨基酸替换中，Ile37突变对蛋白质二聚化和病毒复制的影响最大。Leu35和Ile37的双重突变几乎消除了蛋白质二聚体，并对病毒复制产生了最大的负面影响。这些研究表明，Leu35和Ile37是蛋白质二聚化最重要的残基，对病毒复制至关重要。我们的研究结果还表明，通过破坏Rep或Rep '在N端的二聚化可以减少PCV的复制，这表明负责二聚化的结构界面提供了一个有希望的抗病毒靶点。猪圆环病毒2型（PCV2）是影响养猪业最具经济破坏性的病原体之一。尽管疫苗已问世10多年，但该病毒仍然普遍存在。显然需要更有效的疾病预防战略。病毒的Rep和Rep蛋白具有相同的n端区域，相互作用，允许形成同源或异源二聚体。异二聚体在病毒复制的不同阶段起着至关重要的作用。在这里，我们解析了Rep （Rep’）二聚化结构域的晶体结构。参与分子间相互作用的单个残基在蛋白质结构中可视化，并通过突变体分析验证了几种相互作用。我们的研究表明，破坏相互作用减少病毒复制，从而揭示了抗病毒药物设计的新目标。
Two replicase (Rep) proteins, Rep and Rep′, are encoded by porcine circovirus (PCV) ORF1; Rep is a full ORF1 transcript, and Rep′ is a truncated transcript generated by splicing. These two proteins are crucial for the rolling-circle replication (RCR) of PCV. The N-terminal sequences of Rep and Rep′ are identical and interact to form homo- or heterodimers. The three types of dimers perform different functions during replication. A structural examination of the interfacing termini has not been performed. In this study, a crystal structure of dimerized Rep protein N termini was resolved at 2.7 Å. The dimerized protein was maintained by nine intermolecular hydrogen bonds and 15 pairs of hydrophobic interactions. The amino acid residue Ile37 participates in 11 of the hydrophobic interactions, mostly with its side chain. To find the predominant sites for protein dimerization and virus replication, a series of mutant proteins and virus replicons were generated by alanine substitution. Of all the single amino acid substitutions, the mutation at Ile37 showed the greatest effect on protein dimerization and virus replication. A double mutation at Leu35 and Ile37 almost eliminated protein dimerization and had the greatest negative effect on virus replication. These studies demonstrate that Leu35 and Ile37 are the most important residues for protein dimerization and are crucial for virus replication. Our results also show that PCV replication can be decreased by disrupting the dimerization of Rep or Rep′ at the N terminus, suggesting that the structural interface responsible for dimerization offers a promising antiviral target.IMPORTANCEPorcine circovirus type 2 (PCV2) is one of the most economically damaging pathogens affecting the swine industry. Although vaccines have been available for more than 10 years, the virus still remains prevalent. More effective strategies for disease prevention are clearly required. The Rep and Rep′ proteins of the virus have identical N-terminal regions that interact with each other, allowing the formation of homo- or heterodimers. The heterodimer has crucial functions during different stages of viral replication. Here, we resolved the crystal structure of the Rep (Rep′) dimerization domain. The individual residues involved in the intermolecular interaction were visualized in the protein structure, and several interactions were verified by mutant analysis. Our studies show that disrupting the interaction decreases viral replication, thus revealing a new target for the design of antiviral agents.